Not applicable.
In the art of plant genetics, it has now become common practice to create genetically engineered plants, referred to as transgenic plants, which have stably inserted into their chromosomes one or more foreign gene constructions intended to express a novel or foreign protein in the transgenic plants. Techniques exist to insert genes into plant cells and to regenerate whole fertile transgenic plants from such cells. For several important commercial crop species, transgenic seeds are commercially available and are widely planted and harvested.
The most common techniques currently used for creating plant transformation vectors for plant transformations are based on manipulation and construction of the genetic material in bacterial cells followed by the transfer of the genetic materials from the bacterial cells into plant cells. As most commonly practiced, DNA incorporating a protein coding region for the protein of interest is inserted into a plant expression vector which usually includes a promoter and a transcription termination, or polyadenylation sequence, both of which work in plant cells. The combination of a promoter, protein coding sequence, and a polyadenylation sequence is referred to here as a chimeric gene construction or a plant expression cassette. The plant expression vector often also includes a selectable marker gene, or a gene that confers resistance to a chemical selection agent such as an antibiotic or herbicide. Use of such a selectable marker permits transformed plant cells to be selected from among non-transformed plant cells due to the ability of the transformed plant cells to withstand application of the chemical selection agent to the cells.
Sometimes it is desired that a transgenic plant be constructed that carries more than one foreign gene construction in its genome for more than one gene of interest. If plants are to be altered in their fundamental biochemical characteristics, insertion or alteration of a cascade of enzymes may be needed. While it is possible to incorporate more than one expression cassette into the same plant expression vector, doing so is often not easy or convenient. If two gene cassettes are in the same vector and each includes the same plant promoter or the same polyadenylation sequence, a phenomenon called homologous recombination can occur in the bacterial host which can result in deletion of all of the DNA in the vector which lies in between the two copies of the same promoter. Because there are a limited number of plant promoters known to the art, and since each promoter has different expression characteristics, using two promoters for difference gene cassettes in the same vector can result in different patterns of gene expression for genes that are intended to work in tandem. An alternative approach is to genetically engineer a plant to carry a first inserted gene, called then a transgene, and then to re-engineer the engineered plant to receive a second genetic construction. This approach also has some limitations. First, there are only a few selectable markers known, and one cannot use as a selectable marker in a transformation protocol a marker for which the plant is already resistant. Secondly, when such plant expression cassettes are integrated into the DNA of the plant genome, in general the insertion is at a random location. This gives rise to the so-called xe2x80x9cposition effect,xe2x80x9d which is a poorly defined and poorly understood, but widely observed, phenomenon by which the same gene will express at dramatically different levels in different transgenic plants which have the gene inserted in different locations in its genome. Thirdly, the two inserted genes would randomly insert into separate locations in the plant genome, and would thus be genetically unlinked, complicating transfer of the two genes in tandem during plant breeding. Regardless of whether the two inserted genes are introduced at the same time or at two separate times, two genes containing similar sequences in the promoter or polyadenylation signal can adversely affect the activity of each other leading to suppression of both genes, an effect known as co-suppression or gene silencing.
If one wants to engineer a plant to receive a series of enzymes in a cascade intended to produce an end product, one generally wants the enzymes to be produced at relatively similar levels in the cells of the plant. The prior art does not describe convenient methods for achieving such multiple gene expression in plants with comparable or controllable levels of expression among the inserted genes.
Ubiquitin is an abundant protein of 76 amino acids which is present in the cytoplasm and nucleus of all eukaryotic organisms, including plants. Ubiquitin functions as the central component of the ubiquitin-dependent proteolytic pathway, a principle mechanism of amino acid recycling in cells. Ubiquitin is highly conserved and the amino acid sequence of ubiquitin is invariant in all plant species examined so far. Ubiquitin is naturally synthesized as a part of protein fusions which can be polyubiquitins, consisting of multiple ubiquitin monomers in tandem, or ubiquitin extension proteins in which ubiquitin monomers are linked to the amino-terminus of unrelated proteins. The initial fusion proteins produced from such synthesis are naturally processed in vivo to release functional ubiquitin monomers and functional extensions. The enzymes responsible for this activity are known as ubiquitin-specific proteases or UBPs. UBPs are highly specific for ubiquitin and will remove almost any peptide appended by a peptide bond to the carboxyl terminus of ubiquitin, except when proline is the first amino acid of the linked protein at the carboxyl terminus.
Because of its highly stable structure and the natural occurrence of ubiquitin fusions, fusions based on ubiquitin have been previously described as a method to augment the accumulation of unstable or poorly expressed proteins in plants. U.S. Pat. No. 5,773,705 describes a system for using a ubiquitin fusion protein strategy for improving the expression of some proteins in plants.
The present invention is summarized in that a transgenic plant includes an artificial genetic construction which includes, 5xe2x80x2 to 3xe2x80x2, a promoter operable in plants, a protein coding sequence, and a polyadenylation sequence. The protein coding sequence encodes the expression of a chimeric fusion protein which includes the complete amino acid sequence of at least two proteins of interest joined by a ubiquitin linking domain, the ubiquitin linking domain is cleaved under normal physiological conditions in plant cells to release the two proteins of interest.
The present invention is also summarized in a method for making transgenic plants which includes the steps of constructing a plant gene expression cassette including a promoter operable in plants and a polyadenylation sequence operable in plants, the promoter and the polyadenylation sequence operably connected to a protein coding sequence encoding a fusion protein made up of at least two proteins of interest connected by a ubiquitin linking domain, and transforming the plant gene cassette into a plant such that progeny of the plant produces the two proteins of interest in stoichiometric levels.
It is another aspect of the present invention in that a method is described for the expression of multiple proteins in stoichiometric levels in transgenic plants.
Other object advantages and features will become apparent from the following specification when taken in conjunction with the accompanying drawings.